The long-term goal of the proposed research is to gain a thorough understanding of the physical, biochemical, kinetic and mechanistic properties of the bacterial flavocytochrome, p-cresol methylhydroxylase (PCMH). The primary goal will be to study important aspects of the assembly of PCMH, particularly those relating to the mechanism of covalent FAD attachment to Tyr384 in the flavoprotein subunit. Our second goal will be to study some aspects of substrate binding and the catalytic mechanism, particularly with the modified forms of PCMH and PchF made available for the primary goal. Adjunct studies of goals 1 & 2 will provide information concerning the mechanism of electron transfer from FAD to heme in PCMH. These investigations will involve pure site-specifically altered forms of the PCMH flavoprotein subunit, and pure forms of the wild-type subunit that contain altered flavin cofactors. These unnatural proteins will be studied by a variety of physical, chemical, and kinetic methods. Each of these proteins is designed to test the participation of a particular amino acid, or the property of the flavin in fundamentally important processes of PCMH. This research will be guided by the high-resolution x-ray crystal structures of fully and partially assembled forms of PCMH. PCMH provides a simple, convenient model for oxidoreductases, and specifically for human enzymes that contain covalently bound flavin as cofactors. These include the human mitochondrial enzymes succinate dehydrogenase, monoamine oxidase, dimethylgycine dehydrogenase and sarcosine dehydrogenase, and the human peroxisomal enzymes L-pipecolic acid oxidase and sarcosine oxidase. Information gathered from the studies of PCMH will provide insights into how the human enzymes assemble and function. This information may lead to the development of better treatments for some human afflictions, such as Parkinsonisn, depression, stress, mitochondrial myopathies, and aging.